1. Volume 33, Issue 4, Pages 597-606 (October 2010)
Mammalian Target of Rapamycin Controls Dendritic Cell Development Downstream of Flt3 Ligand Signaling 
Taheri Sathaliyawala, William E. O'Gorman, Melanie Greter, Milena Bogunovic, Vjollca Konjufca, Z. Esther Hou, Garry P. Nolan, Mark J. Miller, Miriam Merad, Boris Reizis 
Immunity 
Volume 33, Issue 4, Pages (October 2010)
DOI: /j.immuni
Copyright © 2010 Elsevier Inc. Terms and Conditions

2. Figure 1 Rapamycin Inhibits Flt3L-Driven DC Development In Vitro
(A) The effect of rapamycin on DC development in vitro. Wild-type BM was cultured in the presence of Flt3L with or without 10 ng/ml rapamycin and analyzed by flow cytometry on day 8 for the resulting pDCs (CD11c+ B220+ CD11b−), CD8−-like cDCs (CD11c+ B220− CD11bhi) and CD8+-like cDCs (CD11c+ B220− CD11blo). Absolute cell numbers per 2 × 106 input BM cells (mean ± SD of three independent cultures) are shown; ∗p < 0.05; ∗∗p < 0.01.
(B) The effect of rapamycin on DC progenitor expansion. CFSE-labeled BM cells were cultured with Flt3L with or without rapamycin and analyzed on day 3. Highlighted are CFSE-diluting pro-DCs with high or low CD43 expression (upper panels) and CFSElo CD11c+ pre-DCs (lower panels), with average percentages ± range of two independent cultures.
(C) The effect of rapamycin at low doses. BM cells were cultured with Flt3L and the indicated doses of rapamycin. Shown are staining profiles on day 8, highlighting CD11c+ B220+ pDCs (top) and CD24+ CD11blo CD8+-like cDCs among the gated CD11c+ MHC II+ cDCs (bottom). Average percentages ± SD of three independent cultures are indicated.
(D) Time-dependent rapamycin activity in Flt3L-supplemented BM cultures. Rapamycin (10 ng/ml) was added at the indicated days, and the cultures were analyzed on day 9. A representative of four independent cultures is shown.
Immunity  , DOI: ( /j.immuni )
Copyright © 2010 Elsevier Inc. Terms and Conditions

3. Figure 2 Pten Deletion in the BM Facilitates DC Development
Global Pten deletion was induced by tamoxifen administration to Ptenfl/flGt(ROSA)26Sor-CreER+ animals (PtenΔ) or littermate controls (Ctrl) 5 days prior to BM isolation.
(A) DC development in Flt3L-supplemented BM cultures on day 5. Shown are staining profiles representative of two independent experiments, with total live cells (forward versus side scatter gate), CD11c+ B220+ pDCs, CD11c+ B220− cDCs and their subsets highlighted.
(B) DC development in Flt3L-supplemented BM cultures at the endpoint. Cultures described in (A) were analyzed on days 8–9. Shown are representative staining profiles of total live cells, and fold increase of the absolute DC numbers in PtenΔ over control cultures (three independent experiments).
(C) DC development in vivo in the BM chimeras. BM cells from each tamoxifen-treated animal (one control and three PtenΔ) were transferred into two irradiated recipients, and splenic DCs were analyzed 6 weeks thereafter. Shown are representative staining profiles of donor-derived (CD45.2+) DCs, and the fractions of cDCs (CD11chi MHC II+ CD8+ or CD8−) and pDCs (CD11clo B220+ Bst2+) among the total donor-derived splenocytes.
Immunity  , DOI: ( /j.immuni )
Copyright © 2010 Elsevier Inc. Terms and Conditions

4. Figure 3 mTOR Signaling in DCs In Vivo
(A) The expression of phosphorylated S6 protein (p-S6) in the splenocytes of naive wild-type mice. Shown is the definition of cDC subsets in ex vivo splenocytes after instant fixation, and p-S6 expression in T cells (CD90+), B cells (B220+ CD11c−), monocytes or macrophages (M, CD11blo CD11c−), and cDCs. Positive staining threshold is indicated by the dotted line.
(B) The induction of p-S6 by Flt3L in vivo. Shown are histograms of intracellular p-S6 fluorescence in the indicated cell types 15 or 60 min after Flt3L administration.
Immunity  , DOI: ( /j.immuni )
Copyright © 2010 Elsevier Inc. Terms and Conditions

5. Figure 4 DC-Specific Pten Deletion Causes the Expansion of CD8+ cDCs
(A) Staining profiles of splenic DC populations in Ptenfl/flItgax-Cre+ animals with DC-specific Pten deletion (DC-PtenΔ) and in littermate controls (Ctrl), highlighting CD11clo Bst2+ pDCs, CD11chi MHC II+ cDCs and their subsets.
(B) The fraction and absolute number of cDC subsets in DC-PtenΔ and control spleens (mean ± SD, n = 10–11). The p values of statistically significant differences are indicated.
(C) The analysis of DCs in hematopoietic chimeras reconstituted with a mixture of the control or DC-PtenΔ BM (CD45.2+) and wild-type CD45.1+ competitor BM. Shown are representative staining profiles and the absolute numbers of donor- and competitor-derived splenic cDC subsets (mean ± SD of three recipient animals).
(D) Splenic cDC subsets in Cx3cr1-EGFP+ DC-PtenΔ and littermate control mice. Shown are staining profiles of CD11chi MHC II+ cDCs with EGFP+ and EGFP- CD8+ cDC subsets highlighted and absolute numbers of cDC subsets (mean ± SD, n = 4–5).
(E) Immature splenic cDC populations in DC-PtenΔ and control mice. Shown are staining profiles of gated MHC II+ CD24+ or CD24− populations containing mature CD11chi cDC subsets as well as CD11clo CD8lo immature cDCs (mean ± SD of three animals).
Immunity  , DOI: ( /j.immuni )
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6. Figure 5 The Expansion of CD103+ cDCs after Pten Deletion
(A) The CD103+ versus CD11b+ cDC subset distribution in the tissues of DC-specific DC-PtenΔ mice. Shown are representative staining profiles of DAPI− CD45+ CD11chi MHC II+ cDCs from the indicated tissues of Cx3cr1-EGFP+ DC-PtenΔ and control mice. The populations of CD103+ and CD11b+ or Cx3cr1-EGFP+ DCs are highlighted; the intestinal lamina propria (LP) contains an additional CD103+ CD11b+ population.
(B) Absolute numbers of CD103+ cDCs in individual DC-PtenΔ mice shown by pairwise comparison to the corresponding littermate control.
(C) The ratio of CD103+ CD11b− to CD103− CD11b+ fractions among cDCs from control and DC-PtenΔ mice (mean ± SEM of 8–9 animals for all tissues except intestinal LP, for which n = 3).
Immunity  , DOI: ( /j.immuni )
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7. Figure 6 mTOR Blockade Reverses the Expansion of Pten-Deficient DCs
(A) Rapamycin treatment of Flt3L-supplemented BM cultures from DC-PtenΔ and control mice. Rapamycin was added on day 3 of culture. Shown are staining profiles of gated CD11c+ B220− cDCs highlighting the CD11blo CD24hi CD8+ cDC-like subset (mean ± range of two independent cultures).
(B) Rapamycin treatment of DC-PtenΔ and control animals. Shown are representative staining profiles of splenic CD11chi MHC II+ cDCs from mice treated for 7 days with rapamycin or vehicle only.
(C) Absolute numbers of splenic cDC subsets from DC-PtenΔ and control animals treated with rapamycin (mean ± SD of 5–6 animals per group).
Immunity  , DOI: ( /j.immuni )
Copyright © 2010 Elsevier Inc. Terms and Conditions

8. Figure 7
DC-Specific Pten Deletion Increases Sensitivity to Listeria Infection
(A) Spleen histology of control and DC-PtenΔ mice on day 6 after infection with 2 × 104 LM-OVA bacteria (H&E staining, 25× magnification; inset, 400× magnification). Uninfected control or DC-PtenΔ mice showed no difference by histology (not shown).
(B) Bacterial titers on day 6 after infection with 2 × 105 LM-OVA. All DC-PtenΔ mice were moribund and showed prominent inflammation of the spleen and liver. Symbols represent individual animals; N.D., not detected.
(C) Immunohistochemical analysis of the spleen 24 hr after infection with 105 LM-OVA. Frozen spleen sections were stained for polymerized actin, CD11c, and ovalbumin for LM-OVA detection.
Immunity  , DOI: ( /j.immuni )
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